Traditionally, drug development has not been based on genetic information. More rational approaches are currently possible, however, based on accumulated knowledge regarding the molecular mechanisms of infectious particles (viruses, bacteria, yeast, fungi and protozoa) and the target sites for antibiotics on the molecular level. Two new approaches which have promise for a more rational drug design are combinatorial chemistry (Gordon, E. M., et. al., J. Med. Chem., 1994, 37, 1385-1401; Alper, J., Science, 1994, 264, 1399-1401) and antisense (Cohen, J. S., et al., Scientific American international edition, December 1994, pages 50-55).
In combinatorial chemistry, a large number of all variants of a specific family of compounds is synthesized and investigated for specific affinity to targeted modules i.e. receptor binding sites. The antisense approach utilizes suitably modified oligonucleotide sequences, which are designed to bind to essential regions for gene expression or virus or cellular replication resulting in complete suppression of the encoded functions.
H-phosphonate or phosphoramidite chemistries employing solid phase methods in automated DNA synthesizers are most efficient for the synthesis of oligonucleotides. The phosphoramidite method using B-cyanoethyl phosphoramidites as reactive nucleotide building blocks is the most prevalent synthesis method due to the quantitative condensation yields despite an oxidation step in every cycle (Sinha, N. D. et al., Tetrahedron Lett., 1983, 24, 5843-46; Sinha, N. D. et al., Nucleic Acids Res., 1984, 125 4539-57; Froehler, B. C. et al., Nucleic Acids Res., 1984, 14, 5399-5407; Froehler, B. C. and Matteuci, M.D., Tetrahedron Left, 1986, 27, 469-72; Garegg, P. J. et at, Tetrahedron Left, 1986, 27, 4051-54; Sonveaux, E., Bioorg. Chem., 1986, 14, 274-325; Uhimann, E. and Peyman, A., Chem. Rev., 1990, 90, 543-84).
According to this method, DNA is synthesized typically in the 3xe2x80x2-5xe2x80x2-direction by using temporary acid labile 4,4xe2x80x2-dimethoxytrityl (DMTr) groups. The base (acyl amide bonds) and phosphate protection (xcex2-cyanoethylxe2x80x94deprotected via xcex2-elimination) and the ester linkage to the support are cleaved in a single step by a nonselective reaction with concentrated aqueous ammonia.
To be useful as drugs, oligonucleotides must be able to penetrate through cell walls and nuclear membranes without undergoing enzymatic degradation. Unmodified oligonucleotides are generally unsuitable for this purpose. Therefore the development of modified oligonucleotides is essential for the antisense/triplex DNA approach. Various modifications have been introduced which mainly alter the internucleotide bond (i.e. methyl phosphonates, phosphorothioates and -dithioates, phosphate triesters, phosphoamidates, replacement of the internucleotide bond involving non-phosphorus containing moieties such as PNAs), the base, 2xe2x80x2-deoxyribose or linkage of various molecules at the 3xe2x80x2- or 5xe2x80x2-OH end of the oligonucleotide (Uhlmann, E. and Peyman, A., Chem Rev., 1990, 90, 543-84; Nielsen, P. et. al., Science, 1991, 254, 1497; Beaucage, S. L. and Iyer, R. P., Tetrahedron., 1993, 49, 6123; Nielsen, P. et. al., Nucleic Acids Res., 1994, 22, 703-10).
Standard synthetic procedures typically result in depurination by the removal of the DMTr group in each elongation cycle (Shabarova, Z., Bogdanov, A. in Advanced Organic Chemistry of Nucleic Acids, VCH Verlagsgesellschaft Weinheim, Germany, 1994). In addition, since the synthesis usually is 3xe2x80x2-5xe2x80x2-directed. oligonucleotides substituted at their 3xe2x80x2-OH end are not easily available. Further, nonselective deprotection by ammonia is disadvantageous for the synthesis of modified DNA, if protecting groups are part of the modification strategy of oligonucleotides.
Antisense/triplex oligonucleotides have special requirements. The hybridization must be specific and strong enough to guarantee a sufficient blocking of mRNA or nuclear DNA target sequences. In addition, the oligonucleotides should be modified to protect against enzymatic degradation (e.g. by exo- and endonucleases) and to facilitate the passage through the cytoplasmic membrane (to access mRNA sequences) and the nuclear membrane (to target DNA sequences by forming triple helices). To be of therapeutic value, obviously, the modified oligonucleotides must also be non-toxic and the synthetic process must be amenable to easy and cost-effective upscaling.
Whether the target sequences of mRNAs are available for hybridization (i.e. located in loops or single stranded areas and not hidden in stem or tertiary structures) cannot be predicted with absolute certainty (Engels, J., Natur. Chem. Techn. Lab., 1991, 39, 1250-54). In addition, a synthetic DNA may also bind to unexpected targets such as proteins (Cohen, J. S. et. al., Scientific American, international edition, December 1994, pages 50-55) as observed in tissue culture treated with phosphorothioate oligonucleotides. This could lead to further requirements and fine tuning for the modification, since improvements in one aspect may cause a disadvantage in another. For example the introduction of polycyclic aromatic compounds can lead to higher affinity for the complementary strand due to the intercalating properties but at the same time reduced specificity could result in an increased toxicity or mutagenicity (Engels, J., Nachr, Chem. Techn. Lab., 1991, 39, 1250-54). In the triple helix approach there is a demand for special structures if the target sequences do not consist of a continuous stretch of purine residues which is a prerequisite for triple helix formation (Cohen, J. S., et. al., Scientific American international edition, December 1994, pages 50-55).
Progress in the syntheses of modified oligonucleotides is remarkable but only a few of the requirements can be fulfilled in one synthesis process since all available procedures lack versatility. Solid phase oligonucleotide synthesis using e.g. monomeric phosphodimorpholino amidites permits the creation of a variety of oligonucleotide phosphate triesters (Uznanski, B. et al., Tetrahedron Lett., 1987, 28, 3401-04). However, the diversity of modification is limited to derivatizations of the phosphate moiety alone. In another example insertion of (R)- and (S)-3xe2x80x2,4xe2x80x2-seco-thymidine in oligodeoxynucleotides (modification of the 2-deoxyribose) (Nielsen, P., et. al., Nucleic Acids Res., 1994, 22, 703-10) resulted only in oligomers with good hybridization properties and stability against 3xe2x80x2-exonuclease degradation. All current synthetic methodologies and strategies are hampered by limited versatility and flexibility since the introduction of each modification requires a separate oligonucleotide synthesis run. The development of optimized modification schemes is therefore time consuming and costly.
There is a tremendous demand for synthetic strategies and methodologies which allow the generation of an almost unlimited amount of sequence specific modifications which can be obtained from one synthesis run. In addition, generation of combinatorial libraries of the same oligonucleotide sequence in various states of protection and/or modification allows the selection of molecules with affinities to non-nucleic acid molecules such as receptor sites by using the oligonucleotide backbone as an oligomeric scaffold exhibiting different patterns of functionalities available for specific molecular recognition processes.
The problems discussed above, which can be overcome by this invention, can be summarized as follows:
1) Using new protection schemes and solid phase synthesis, oligonuclec tides are obtained in 5xe2x80x2 to 3xe2x80x2 direction using phosphoamidites and avoiding depurination. The 3xe2x80x2-OH protecting group employed is suitable as a purification handle for HPLC purification and can be detected in the visible spectral region with high sensitivity to determine condensation yields.
2) Each protecting group of the oligonucleotide (inclusive of the support-spacer moiety at the 5xe2x80x2- or 3xe2x80x2-OH end of the oligonucleotide) is removable selectively (multiselectivity of deprotection, principle of orthogonality). Furthermore, phosphate and base protecting groups can be removed and selected at preprogrammed positions. By substitution reactions, a simple and rapid synthesis of a variety of derivatives for the antisense/triplex concept can be made available. All possible derivatizations can be performed with only one oligonucleotide synthesis run. This versatile synthesis method is therefore amenable to scale-up.
3) Furthermore, the selective and orthogonal procedures and derivations can be applied to structures other than oligonucleotides. Thereby generating variously modified molecules to be tested for interactions with a specific molecular target or biological recognition process.
The above and further features and advantages of the instant invention will become clearer from the following Detailed Description and Claims.